As part of the work for Consortium 3, we have been identifying Nap1 binding proteins using two dimensional gel electrophoresis and mass spectrometry. Samples from a Nap1 affinity column were TCA precipitated, solubilized, and sent to the Resource Center by the Kellogg lab. We fractionated one of the aliquots on a two-dimensional gel, and found rather poor resolution of many proteins, possibly due to protein aggregation. With the remaining aliquots (6), we attempted a number of protocols aimed at better resolving the proteins, especially in the first dimension (isoelectric focusing). We were successful in improving the resolution of the separated proteins, and have started with the in-gel digestion and identification of the Nap1 binding proteins using mass spectrometry.